TECHNICAL BRIEF Critical Point Drying

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1 TECHNICAL BRIEF Critical Point Drying Document Number Issue 2 Page 1 of 9 Critical Point Drying Technical Brief November 2009

2 Quorum Technologies Ltd main sales office: South Stour Avenue Ashford Kent U.K. Tel: ++44(0) TN23 7RS Fax: ++44(0) Website: For further information regarding any of the other products designed and manufactured by Quorum Technologies, contact your local representative or directly to Quorum Technologies at the address above. Carbon and sputter coaters Plasma reactor for ashing and etching High vacuum bench top evaporators Cryo-SEM preparation systems Critical point dryers Freeze dryers for electron microscopy Service and Spares Disclaimer The components and packages described in this document are mutually compatible and guaranteed to meet or exceed the published performance specifications. No performance guarantees, however, can be given in circumstances where these component packages are used in conjunction with equipment supplied by companies other than Quorum Technologies. Page 2 of 9 Critical Point Drying Technical Brief November 2009

3 CRITICAL POINT DRYING INTRODUCTION The advent of Scanning Electron Microscopy (SEM) in the study of surface morphology in biological applications made it imperative that the surface detail of a specimen was preserved. Air (evaporative) drying of specimens can cause severe deformation and collapse of structure - the primary cause of such damage being the effects of surface tension. The specimen is subject to considerable forces, which are present at the phase boundary as the liquid evaporates. The most common specimen medium, water, has a high surface tension to air; by comparison that for acetone is considerably lower. The surface tension could be reduced by substitution of a liquid with a lower surface tension with thereby reduced damage during air-drying. However, the occurrence of what is known as 'continuity of state' suggests a drying technique for which the surface tension can be reduced to zero. If the temperature of liquefied gas is increased the meniscus becomes flatter indicating a reduction in the surface tension. If the surface tension becomes very small the liquid surface becomes very unsteady and ultimately disappears. When this 'critical point' is reached, it is possible to pass from liquid to gas without any abrupt change in state. If a specimen had been in the liquid during this process it would have experienced a transition from a wet to a 'dry' gas environment without being in contact with a surface, in this way avoiding the damaging effects of surface tension. This is termed Critical Point Drying (CPD), the basis of which are the classic experiments carried out over 100 years ago during investigations on the liquification of gases. THE CRITICAL PHENOMENA The principle of the experiments, which were initially carried out using carbon dioxide (CO 2 ), was to measure the change in volume with the application of pressure, of a fixed mass of gas, while maintaining a constant temperature. This was repeated for a range of different temperatures. The results are best understood by considering the graph obtained from plotting pressure (P) against volume (V) for the series. This is shown in Figure 1; the curves obtained are termed isothermals Page 3 of 9 Critical Point Drying Technical Brief November 2009

4 Figure I Consider first the 10 o C isothermal at low applied pressure. The CO 2 is gaseous (vapour) and generally exhibits the characteristics of a gas (Boyle s Law) over the range from 'r' to 's'. From point 's' a very slight increase in pressure results in a change from vapour state to the liquid state. This is the phenomena of saturation. From 's' to 't' the pressure is virtually constant while the volume is decreasing and at 't' the substance is all liquid. From point 't' the graph becomes almost vertical indicating significant application of pressures for very little change in volume, liquids being virtually incompressible. The 20 o C isothermal has similar general characteristics, however there is less difference between points 'v' to 'w' compared to the difference between equivalent points 's' to 't' on the 10 o C isothermal; these points representing the difference in volume occupied between the vapour phase and the liquid phase. This indicates that the densities of the saturated vapour and liquid are approaching each other, also the slight departure from the vertical 'w' shows the compressibility is greater than that at higher pressures. This shows that the properties of the liquid and gas states of the substance are becoming similar and will ultimately coincide. This in fact is realized at the 31.1 o C isothermal, which does not show any horizontal discontinuity. The temperature at which this occurs is termed the Critical Temperature and has an associated Critical Pressure Page 4 of 9 Critical Point Drying Technical Brief November 2009

5 and density and hence for a particular mass of gas, a Critical Volume. If a liquid was heated in a closed system so that the critical pressure could be attained, at the critical temperature, any visible meniscus would disappear; the surface tension would be zero and it would not be possible to distinguish between the properties of a liquid or a gas. We therefore have continuity of state. Above this temperature the gas cannot be liquified by the addition of pressure and strictly speaking a substance should only be classified as a gas above its critical temperature, below this temperature where it could possibly be liquified by the application of pressure, it is more precisely termed a vapour. CRITICAL POINT DRYING (CPD) The critical phenomena can be utilized as a drying technique as it achieves a phase change from liquid to dry gas without the effects of surface tension and is therefore suitable for delicate biological specimens. However, it is not surprising that the initial investigations were on CO 2 as will be apparent from Figure 2, showing a table of critical constants for some common substances. Even the practical achievement of the critical conditions would not assist the biologist, as the specimens would suffer significant thermal damage if we attempted to apply the technique direct for the removal of water from specimens. CRITICAL CONSTANTS Substance Temp. o C P.S.I. Hydrogen Oxygen Nitrogen Carbon dioxide Carbon monoxide Water Figure 2 Therefore CO 2 remains the most common medium for the CPD procedure and is termed the 'Transitional Fluid'. However, CO 2 is not miscible with water and therefore water has to be replaced in the specimen with another fluid which is miscible with CO 2, this is termed the 'Intermediate Fluid'. Ideally it will be able to replace the water in the specimen, and also serve as the 'Dehydration Fluid'. This is not exclusively the case, and additional steps may be used for particular circumstances. However, where it is being utilised for both processes, texts may refer to it under the different headings, dehydration and intermediate, depending at what stage it is being used in the specimen preparation schedule. Prior to any of these stages chemical fixation of the specimen must be carried out (normally using glutaraldehyde -osmium procedures). Page 5 of 9 Critical Point Drying Technical Brief November 2009

6 NOTE The whole discipline of specimen preparation (chemical or vapour fixation) prior to the transitional stage is only mentioned in its most basic terms, procedures vary according to the type and nature of the specimens. Further references should be obtained. Critical point drying stages a) Intermediate Stage As mentioned previously this involves dehydration and intermediate fluids, the following is a possible schedule. (Wet Specimen) H 2 O -> Acetone -> C 2 O -> CPD (Dry Specimen) The specimen is usually processed through varying concentrations of dehydration fluid, culminating in complete replacement of the water with this intermediate fluid. Because it has a low surface tension the specimen is less likely to experience damage due to evaporation while transferring to the chamber, also being miscible with CO 2 (the Transitional Fluid) ensures satisfactory conditions after flushing (purging) for the CPD process to commence. (Wet Specimen) H 2 O -> Acetone -> 30%* 100% ->C02**->CPD (Dry Specimen) Page 6 of 9 Critical Point Drying Technical Brief November 2009

7 Note: 50/60/70/80/90 typically 10 minutes each ** Flush Typically 3 times The table (Figure 3) gives an indication of some intermediate fluids. (Water is 73 Dynes/cm.) DEHYDRATION/INTERMEDIATE FLUIDS FOR C.P.D. SUBSTANCE SURFACE TENSION (DYNES/CM) ETHANOL 23 ACETONE 24 FREON (113) 19 WATER 73 Figure 3 Having transferred the specimen to the chamber in the intermediate fluid, the chamber is flushed several times to replace it with the transitional fluid. The critical point drying process can now be started. (b) Transitional Stage As discussed previously (see Figure 1) the conditions for which the critical point passage can be obtained for CO 2 are 31.1 C and 1072 psi. However, it must be remembered that these isothermals are obtained from a fixed mass of gas and an applied pressure for a series of constant temperatures. In the laboratory application of CPD we have a fixed volume which is filled with the transitional fluid. Some typical examples of which are given in figure 4. TRANSITIONAL FLUIDS FOR CPD SUBSTANCE CARBON DIOXIDE FREON 13 FREON 23 WATER CRITICAL TEMP O C PSI Figure 4 Pressure is obtained by the effect of applying heat and while it can be readily appreciated that we can take a liquid from below its critical temperature and obtain the transition to gas above its critical temperature, an understanding of the relevant 'start' and 'end' points and the cycle involved is required in Page 7 of 9 Critical Point Drying Technical Brief November 2009

8 evaluating the design and performance of CPD equipment. It is still useful however, to utilise these CO 2 isothermals as indicated in figure 5 with the Superimposed arrows' showing differing conditions for the CPD device. Figure 5 It is already acknowledged that these circumstances are not exactly comparable. For example, during operation of the CPD we would fill at CO 2 cylinder pressure and at ambient temperature: not at saturated vapour pressure. At a lower temperature decompression is as a result of venting and the subsequent reduction in mass of gas, not reduction in externally applied pressure. The relative volume is determined by the initial level of liquid in relationship to the total free volume available (this being the chamber plus sample boat etc.) If we consider 'X' with the liquid CO 2 more than half filling the total available volume and we heat from 10 C to 35 C then we will make the transition from liquid to gas. The pressure rise will be rapid as the liquid will expand and the level will increase before the critical temperature is reached. This is termed 'going around' the critical point. Usually (as in the case of instruments supplied by Quorum Technologies) a pressure-bursting disc is employed to prevent excessive pressure increase. For condition 'Y' with approximately a full pressure chamber, the liquid level will remain relatively constant, its density decreasing and that of the vapour increasing, and becoming the same when its critical temperature has been reached, together with the corresponding critical pressure. Looking at condition 'Z' with the pressure chamber less than half full. The level will fall and vaporization will occur before the critical temperature is reached, also the specimens may be uncovered and subjected to unwanted evaporation. Page 8 of 9 Critical Point Drying Technical Brief November 2009

9 Ideally, we wish have a situation where the liquid fills the specimen chamber, while still only accounting for approximately 50% of the total volume available. This will ensure that specimens are not uncovered during initial flushing stages and in addition this should enable critical constants of temperature, pressure, and density to be achieved relatively simultaneously without excessive pressure or evaporation conditions occurring. It is also advisable to maintain a temperature somewhat above the critical temperature during decompression, this will avoid the possibility of gas recondensing. It is also important to control the decompression rate itself as there is evidence that rapid pressure equalization can cause specimen. Page 9 of 9 Critical Point Drying Technical Brief November 2009

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